CHROMATIN STRUCTURE AND STABILITY IN THE SOLUTION STATE

核小体阵列由核心组蛋白八聚体-DNA复合物组成 沿着DNA分子以约200 bp的间隔沿着排列。他们是最基本的 染色体超结构的基石，以及所有 真核细胞核中的功能过程;例如，转录， 复制修复溶液中的核小体阵列处于平衡状态 在未折叠、中度折叠、广泛折叠和寡聚化之间 构象状态这些状态中的每一个的形成都是由 核心组蛋白N-末端，通过多种分子机制起作用。 将接头组蛋白并入核小体阵列以形成染色质 阵列使平衡向广泛折叠和寡聚化的方向移动， 状态，并在此过程中显着改变连接DNA的结构 连接阵列中相邻的核小体。建议的目标 研究的目的是描述和表征核心的机制， 组蛋白N-末端和连接组蛋白对染色质折叠的影响 和通过真核RNA聚合酶的转录。具体来说，使用长度- 和组成确定的核小体和染色质阵列组装， 纯DNA和组蛋白成分，结合创新的技术， 该方法集成了分析超离心、定量琼脂糖 凝胶电泳，电泳，和电子冷冻显微镜，博士。 汉森建议：（1）描述选择性去除和靶向 核心组蛋白N端乙酰化影响核小体阵列折叠 和寡聚化，并确定N-末端是否形成同型或 异型复合物，（2）表征不同接头 组蛋白结构域和序列同种型影响染色质折叠和接头 DNA结构，和（3）进行相关的体外转录研究， 确定染色质折叠抑制RNA转录的程度 聚合酶II和III。所提出的实验是强假设的 驱动，并直接解决几个长期存在的范式， 染色质结构和功能。这些研究将有助于提供 实现功能性组装的长期目标所必需的基础 体外重要遗传位点完全来自纯组蛋白和非组蛋白 件.

Nucleosomal arrays consist of core histone octamer-DNA complexes spaced at ~200 bp intervals along a DNA molecule. They are the fundamental building blocks of chromosomal superstructures, and the substrates for all functional processes in the eukaryotic nucleus; e.g., transciption, replication, repair. Nucleosomal arrays in solution exist in equilibrium between unfolded, moderately folded, extensively folded and oligomerized conformational states. Formation of each of these states is mediated by the core histone N-termini, acting through multiple molecular mechanisms. Incorporation of linker histones into nucleosomal arrays to form chromatin arrays shifts the equilibrium towards the extensively folded and oligomerized states, and in the process dramatically alters the structure of the linker DNA that connects adjacent nucleosomes in the array. The objectives of the proposed research are to delineate and characterize the mechanisms by which the core histone N-termini and linker histones exert their effects on chromatin folding and transcription by eukaryotic RNA polymerases. Specifically, using length- and compositionally-defined nucleosomal and chromatin arrays assembled from pure DNA and histone components, in combination with an innovative technical approach that integrates analytical ultracentrifugation, quanititative agarose gel electrophoresis, and electrophoresis, and electron cryo-microscopy, Dr. Hansen proposes to: (1) characterize how selective removal and targeted acetylation of the core histone N-termini influence nucleosomal array folding and oligomerization, and to determine whether the N-termini form homotypic or heterotypic complexes with each other, (2) characterize how different linker histone domains and sequence isotypes influence chromatin folding and linker DNA structure, and (3) perform correlative in vitro transcription studies to determine the extent to which chromatin folding represses transcription by RNA polymerases II and III. The proposed experiments are strongly hypothesis driven, and directly address several long-standing paradigms relating to chromatin structure and function. These studies will help provide the foundation necessary to achieve the long-term goal of assembling functionally important genetic loci in vitro entirely from pure histone and non-histone components.